EUR 23997 EN - 2009

Evaluation of the Effect of Mycotoxin Binders in Animal Feed on the Analytical Performance of Standardised Methods for the Determination of Mycotoxins in Feed

A. Kolossova, J. Stroka, A. Breidbach, K. Kroeger, M. Ambrosio, K. Bouten, F. Ulberth

The mission of the JRC-IRMM is to promote a common and reliable European measurement system in support of EU policies. European Commission Joint Research Centre Institute for Reference Materials and Measurements Contact information Address: Joerg Stroka, Retieseweg 111, B-2440 Geel E-mail: Joerg.stroka@ec.europa.eu Tel.: +32-14-571229 Fax: +32-14-571783 http://irmm.jrc.ec.europa.eu/ http://www.jrc.ec.europa.eu/ Legal Notice Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of this publication.

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A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server http://europa.eu/ JRC 54375 EUR 23997 EN ISBN 978-92-79-13106-6 ISSN 1018-5593 DOI 10.2787/15352 Luxembourg: Office for Official Publications of the European Communities © European Communities, 2009 Reproduction is authorised provided the source is acknowledged Printed in Belgium

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Table of Contents Summary……………………………………………………………………………………………….4 Background Information on Mycotoxin Binders and Their Use…………………………………...5 Outline of the Study…………………………………………………………………………………..13 Methodology………………………………………………………………………………………..…14 Results and Discussion………………………………………………………………………………..19 Conclusion……………………………………………………………………………………………..26 Acknowledgement…………………………………………………………………………………….27 References……………………………………………………………………………………………..28 Annex 1………………………………………………………………………………………………...34 Annex 2………………………………………………………………………………………………...36

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Summary

The last few years have brought a vast amount of information about mycotoxin inactivation agents

(mycotoxin binders) which are applied with the aim to reduce the toxic effects of mycotoxins in

animals. The influence of the addition of mycotoxin binders to animal feed on the analytical

performance of the methods for the determination of mycotoxins was studied and the results are

presented in this report. Standardised methods already available or currently under consideration at the

European Standardization Committee (CEN) have been applied for the analysis of mycotoxins in feed

materials. Samples of 20 commercial mycotoxin inactivation agents were collected from various

companies. The following mycotoxins were included in the study: aflatoxin B1, deoxynivalenol,

zearalenone, ochratoxin A, fumonisins B1 + B2, T2 and HT2 toxins. Naturally contaminated or spiked

feed materials and the maximum recommended amounts of the mycotoxin detoxifying agents were

used in the experiments. A binder (or binders combined in a group) was mixed with feed material

containing the corresponding mycotoxin, and the feed material with and without binder was analysed

using the appropriate method. For data evaluation, the obtained mean values were compared by

Student’s t-test (independent two-sample t-test with unequal sample sizes and equal variance). The

repeatability standard deviation of each method based on collaborative trial data was used as an

estimate of method variability. No significant differences (p = 0.05) in mycotoxin levels between

binder free material and the material containing different binders have been found under the applied

conditions.

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Background Information on Mycotoxin Binders and Their Use

Mycotoxins are toxic secondary metabolites produced by several fungi, particularly by many

species of Aspergillus, Fusarium, Penicillium, Claviceps, and Alternaria. It has been estimated that at

least 300 of these fungal metabolites are potentially toxic to animals and humans [1]. The most

extensively investigated mycotoxins are aflatoxin B1 (AFB1), ochratoxin A (OTA) zearalenone

(ZEA), deoxynivalenol (DON, “vomitoxin”), T-2 and HT-2 toxins, and fumonisins (FUM).

Mycotoxins are produced by fungi during growth, handling and storage of agricultural commodities.

Their global occurrence is considered to be a major risk factor. Worldwide, approximately 25% of

crops are affected by mycotoxins [2]. The economic consequences of mycotoxin contamination are

profound, and often crops with large amounts of mycotoxin have to be destroyed.

There is an increasing awareness of the hazards posed to both human and animal health by the

presence of toxins produced by fungi in food and feed. Mycotoxins have a diversity of chemical

structures which accounts for different biological effects. They can be carcinogenic, mutagenic,

teratogenic, oestrogenic, neurotoxic, immunotoxic, etc. In farm animals, mycotoxins can cause, among

others, decreased performance, feed refusal, poor feed conversion, diminished body weight gain,

immune suppression, reproductive disorders, and residues in animal food products [1, 3]. However, the

progression and diversity of symptoms are confusing and diagnosis is difficult [4, 5]. Diagnosis is

further complicated by a lack of research, by nonspecific symptoms and by interactions with other

stress factors. Mycotoxin effects are also moderated by a number of factors, such as animal species,

gender, age, diet, and duration of exposure. Many mycotoxigenic fungi can grow and produce their

toxic metabolites under similar conditions. Therefore in animal feed, mycotoxins rarely occur as single

contaminants [6]. Apart from that, blends of various raw materials in compound feed can increase the

risk of feed contamination with several mycotoxins. Several combinations of mycotoxins have been

reported [7-9], such as the co-occurrence of aflatoxin B1 with ochratoxin A or that of deoxynivalenol

with zearalenone, nivalenol, or other Fusarium toxins. Intake of combinations of mycotoxins may lead

to interactive toxic effects. Furthermore, the toxic effect of any single mycotoxin may be amplified due

to synergistic interactions with other substances [9]. Regardless of the difficulty of diagnosis,

mycotoxins should be considered as a possible cause of production and health problems when such

symptoms exist and problems are not attributable to other typical causes [5].

Mycotoxin legislation in the EU

Due to the frequent occurrence of mycotoxins and their severe toxic properties in animals,

legislative limits and recommendations for these compounds have been set for feedstuff in Europe. For

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animal feed, maximum level set by the European Commission (EC) for aflatoxin B1 is 0.02 mg/kg for

all feed materials [10]. For other feeding stuffs, the legal limits vary from 0.005 to 0.02 mg/kg.

Recommendations on the presence of deoxynivalenol, zearalenone, ochratoxin A, fumonisins B1+B2,

T-2 and HT-2 toxins in products intended for animal feed were given by the EC. The corresponding

guidance values for these mycotoxins except for T-2 and HT-2 have been established [11].

Prevention of mycotoxin contamination in the field

Prevention of fungal infections during plant growth, harvest, storage and distribution would

seem the most rational and efficient way to avoid mycotoxins in agricultural commodities [1, 12-14].

Common practical measures include planting of more resistant varieties of cereals, selection of high

quality seeds, avoiding high plant densities, balanced fertilisation, preventive management towards

insect infestations as well as a suitable management of crop residues that are often the primary

inoculum of mycotoxigenic fungi. Careful selection of harvest date, equipment and harvesting

procedures to minimise crop damage and removal of damaged crops and high moisture plant parts also

reduces mould infections. Immediate storage in good storage facilities (moisture, temperature,

humidity and insect control) and the addition of antifungal agents may also diminish fungal growth but

cannot detoxify contaminated feedstuffs. Furthermore, the growth of fungi and therefore, the

production of mycotoxins is limited by the use of propionic acid or ammonium isobutyrate, which can

be used for a post-harvest treatment. Calcium propionate has also been suggested as a mould inhibitor

[15].

Detoxification of animal feed from mycotoxins

Although the prevention of mycotoxin contamination in the field is the main goal of

agricultural and food industries, under certain environmental conditions the contamination of various

commodities with mycotoxins is unavoidable. Feed additives like antioxidants, sulphur containing

amino acids, vitamins, and trace elements can be useful to reduce the toxic effects observed in animals

[16].

The increasing number of reports on the presence of mycotoxins in feeds has given rise to a

demand for practical and economical detoxification procedures. A number of approaches have already

been used to counteract mycotoxins, though only a few have real practical application.

Physical treatment includes washing, polishing, mechanical sorting and separation, density

segregation, flotation, autoclaving, roasting and microwave heating, UV irradiation, ultrasound

treatment and solvent extraction [17]. However, the efficiency of these techniques depends on the level

of contamination and on the distribution of mycotoxins throughout the grain. Additionally, the results

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obtained are uncertain and often connected with high product losses. Moreover, some of these physical

treatments are relatively costly and may remove or destroy essential nutrients in feed.

Chemical methods require not only suitable reaction facilities but also additional treatments

(drying, cleaning) that can make them time consuming and expensive. Nevertheless, various chemicals

including oxidising and reducing agents, acids, bases, salts and chlorinating substances have been

tested for their ability to degrade mycotoxins in agricultural commodities. Only a limited number of

these are effective without diminishing the feed nutritional value or palatability. Chemically, some

mycotoxins can be destroyed with calcium hydroxide, monoethylamine, ozone or ammonia [1, 13, 18,

19]. Particularly, ammoniation is an approved procedure for the detoxication of aflatoxin contaminated

feed in several countries [1]. The average ammoniation costs vary between 5 and 20% of the value of

the commodity [20]. Main drawbacks of this kind of chemical detoxication are the ineffectiveness

against other mycotoxins and the possible deterioration of animal health by excessive residual

ammonia in the feed.

With regard to mycotoxin decontamination, the EC is in favour of the use of physical

decontamination processes and sorting procedures [21]. However, neither the use of chemical

decontamination processes, nor the mixing of batches with the aim of decreasing the level of

contamination below the maximum tolerable level are legal within the European Union [21].

Another way of trying to reduce the uptake of mycotoxins from contaminated feed is the use of

mycotoxin binders. They are added to the feed with the intention to exhibit a completely different

mode of action than the above mentioned physical and chemical treatment. The aim of these additives

is to inhibit the uptake of mycotoxins by an animal in vivo. The use of mycotoxin binding agents is

occasionally recommended to farmers in order to protect animals against the harmful effects of

mycotoxins occurring in contaminated feeds. These adsorbent materials are intended to act like a

‘chemical sponge’ and adsorb mycotoxins in the gastrointestinal tract, thus preventing the uptake and

subsequent distribution to target organs. The efficacy of the adsorption appears to depend on the

chemical structure of both the adsorbent and the mycotoxin. The most important feature for adsorption

is the physical structure of the adsorbent, i.e. the total charge and charge distribution, the size of the

pores and the accessible surface area. On the other hand, the properties of the adsorbed mycotoxins,

like polarity, solubility, shape and charge distribution, also play a significant role [1, 21].

Several studies have shown that a variety of adsorbent materials have high affinity for

mycotoxins by the formation of stable linkages. Examples are activated carbon, hydrated sodium

calcium aluminosilicates (HSCAS) and some polymers. These linkages have also been reported to

occur in several liquid systems such as water, beer, wine, whole and skimmed milk, and peanut oil [1,

3, 22-25]. Many adsorbents have been extensively studied and are promoted as animal feed additives.

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However, most of them appear to bind to only a small group of toxins while showing very little or no

binding to others [1]. For example, HSCAS are quite effective with respect to aflatoxins but they fail

to prevent toxic effects of Fusarium mycotoxins, such as fumonisins, trichothecenes or zearalenone

[21].

Inactivation of mycotoxins by adsorbents has been extensively reviewed elsewhere [1, 17, 21-

24]. Most studies related to the alleviation of mycotoxicosis by the use of adsorbents are focused on

aluminosilicates, mainly zeolites, HSCAS, and aluminosilicate-containing clays, all consisting of

aluminates, silicates and some interchangeable ions, mainly alkali metal and alkaline earth metal ions

[1, 26-29]. Mineral clay products such as bentonites, zeolites, and aluminosilicates are the most

common feed additives which are effective in binding/adsorbing aflatoxins [21]. When saturated with

water, the surfaces of these additives attract polar functional groups of some mycotoxins. This effect

isolates the mycotoxin from the digestive process and is thought to inhibit its absorption [30]. The

mechanism of mycotoxin binding, however, has not been extensively studied. The high affinity of

certain adsorbents for aflatoxin B1 was interpreted as the formation of a complex by the β-carbonyl

system of the aflatoxin molecule with aluminium ions [31]. Selection criteria for the evaluation of

properties of smectite clays influencing the sequestration of aflatoxin have been suggested. The

structural aspects of the interaction between aflatoxin and smectite clay have also been discussed [29].

Hydrated sodium calcium aluminosilicates at 1.0% addition level to the feed was reported to

diminish significantly the adverse effects of aflatoxins in young animals [30]. Aluminosilicates are

also used at a level up to 2% in complete diets as “anti-caking” agents. Thus, the original intention for

the addition is another than mycotoxin binding. These compounds, however, show a number of

disadvantages, not least being the impairment of mineral utilization at levels above 2% and a narrow

range of binding efficacy [1]. In animals, aluminosilicates appear to be selective in their

“chemisorption” of aflatoxins with little or no beneficial effect against zearalenone, fumonisin B1,

ochratoxin A, and trichothecenes, including deoxynivalenol, T-2 toxin, or diacetoxyscirpenol [32].

This limitation can be overcome by the use of chemically modified clays. Modifications consist of

alterations of surface properties by exchange of structural charge-balance cations with high molecular

weight quaternary amines, which results in an increased hydrophobicity [28]. In vitro results have

verified the binding efficacy of modified montmorillonite and clinoptilolite against zearalenone and

ochratoxin A [28, 33]. However, the potential toxicity of some of these clay types was pointed out

[34]. In vitro adsorption of zearalenone by a modified montmorillonite nanocomposite has been

reported [35]. This material demonstrated the ability to bind zearalenone in aqueous solutions with

little nonspecific adsorption of common nutrients, vitamin E and lysine. Zeolites have also been shown

to be efficient against zearalenone toxicosis by some in vivo studies [28].

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Regarding the applicability of aluminosilicates for the binding of mycotoxins, it can be

concluded that available studies recognise their effect in preventing aflatoxicosis, but their efficacy

against zearalenone, ochratoxin, and trichothecenes is limited. In addition to the narrow binding range

concerning different mycotoxins, aluminosilicates have the disadvantage of adsorbing micronutrients

and showing high inclusion rates for vitamins and minerals. The risk of natural clays to be

contaminated with dioxins has also to be considered [1, 17].

Activated carbon can efficiently adsorb most of the mycotoxins in aqueous solution, whereas

different activated charcoals have less or even no effects against mycotoxicosis. This might be due to

the fact that activated charcoal is a relatively unspecific adsorbent and, hence, essential nutrients are

also adsorbed particularly if their concentrations in feed are much higher compared to those of a

mycotoxin [1]. However, activated carbons have shown their efficacy in in vivo studies for aflatoxins,

ochratoxin A, diacetoxyscirpenol, and T-2 toxin [32] and in experiments in vitro using a

gastrointestinal model for deoxynivalenol, nivalenol, and zearalenone [21, 36, 37].

Polymers, such as cholestyramine (an anion exchange resin) and polyvinylpyrrolidone (highly

polar amphoteric polymer), have also been demonstrated to bind mycotoxins in vitro and in vivo [1].

Thus, cholestyramine was proven to be an effective binder for fumonisins and zearalenone in vitro. Its

efficacy was confirmed by experiments in a dynamic gastrointestinal model for zearalenone and by in

vivo experiments for fumonisins [21]. However, the cost of those polymers would be a limiting factor

for practical applications.

Facing the relative inefficacy of the clay binders towards mycotoxins other than aflatoxins,

natural organic binders have been proposed [17]. A novel strategy to control mycotoxicoses in animals

is the application of microorganisms capable of biotransforming certain mycotoxins into less toxic

metabolites. The microorganisms act in the intestinal tract of animals prior to the absorption of the

mycotoxins. Many species of bacteria and fungi have been shown to enzymatically degrade

mycotoxins. However, a question concerning toxicity of products of enzymatic degradation and

undesired effects of fermentation with non-native microorganisms on food quality remains open [38].

Yeasts and lactic acid bacteria (LAB) occur as part of natural microbial population in spontaneous

food fermentation and as starter cultures in the food and beverage industry. Saccharomyces cerevisiae

and LAB, the two most important microorganisms in food fermentation, have been shown to bind

different mycotoxins strongly to cell wall components [17, 38, 39].

Besides its excellent nutritional value, yeast or yeast cell walls show a potential as mycotoxin

binders. Using only yeast cell walls instead of whole cells, the adsorption of mycotoxins can be

enhanced. The cell walls harbouring polysaccharides (glucan, mannan), proteins, and lipids exhibit

numerous different and easily accessible adsorption centres as well as different binding mechanisms,

e.g. hydrogen bonds, ionic, or hydrophobic interactions [1]. It has recently been demonstrated that the

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β-d-glucan fraction of yeast cell wall is directly involved in the binding process with zearalenone, and

that the structural organization of β-d-glucans modulates the binding strength. Hydrogen and Van Der

Waals bonds have been evidenced in the glucans-mycotoxin complexes [17, 38, 40]. Probably, a

similar chemical mechanism is involved in the binding process of mycotoxins by LAB. It appears that

the carbohydrate components are common sites for binding, with different toxins having different

binding sites. It is also known that toxin binding in both S. cerevisiae and LAB is strain dependent

[38].

A bacterial strain, which belongs to the genus Eubacteria, has been originally isolated from

bovine rumen fluid. It was found to have trichothecene detoxifying activity and was named BBSH

797. During its metabolism, BBSH 797 produces enzymes (de-epoxidases) that degrade trichothecenes

by selective cleavage of their 12,13-epoxy group which is important for the toxicity of these

mycotoxins. This detoxification was investigated for several trichothecenes. During its manufacture,

BBSH 797 is stabilised by freeze-drying and embedding in protective substances (mainly organic

polymers), to guarantee sufficient stability against environmental conditions during storage and its

passage through the acidic gastric tract of animals. The mode of action of this strain was demonstrated

in vitro and in vivo [7, 41]. Further, a novel yeast strain capable of degrading ochratoxin A and

zearalenone was isolated and characterized. Due to its property to degrade mycotoxins this strain was

named Trichosporon mycotoxinivorans [41, 42].

The recommended dose for the extracted active yeast compounds is in the range of 1-2 kg/ton

of feed. Organic binders are efficient against a larger range of mycotoxins than inorganic binders,

which makes them more adapted to the most frequent cases of multi-contaminated feeds. Apart from

that, they are biodegradable and do not accumulate in the environment after being excreted by animals.

On the contrary, clays which are incorporated at a higher rate than organic binders, accumulate in

manure and then in the field during spreading and can harm soils and pastures [17].

Various products which combine mycotoxin binding properties of different compounds have

recently been developed to counteract the biological effects of co-occurring mycotoxins in animal

feed. A study to assess the multimycotoxin binding efficacy of a commercial product has been

conducted using a dynamic model simulating the kinetic conditions in the gastrointestinal tract of pigs

[6]. The bioavailability of different classes of mycotoxins commonly co-occurring in animal feedstuffs

has been simultaneously assessed. On the basis of the results, it has been assumed that this product

might significantly decrease in vivo the bioavailable amounts of zearalenone, fumonisins and

ochratoxin A, and almost completely prevent intestinal adsorption of aflatoxins in contaminated feed.

Due to their composition and the presence of different adsorbent materials, the products with

multibinding capacity towards chemically different mycotoxins can be beneficial in reducing both

individual and combined adverse effects of mycotoxins in animals.

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The extensive use of adsorbent materials in the livestock industry has led to the introduction of

a wide range of new products, which still have no legally approved status in the EU market for its

intended purpose as a mycotoxin binder. Before any feed additive is applied to prevent mycotoxin

intoxication, it is essential to establish its reliability and safety while considering the economic

feasibility. Moreover, it should be assured that adsorbent materials do not affect nutritional properties.

Many studies have been performed to evaluate different commercial products intended to be used as

mycotoxin binding agents and various aspects have been discussed [21, 26, 43-50]. Masoero et al. [49]

concluded that physical processing of feed, such as pelleting, had a significant effect on the ability of

added sequestering agent to reduce levels of AFM1 in milk when cattle was fed with AFB1

contaminated feed [49]. It was also suggested that even though the ratio of AFB1 to sequestering agent

in the contaminated feed could be an important variable to consider when using sequestering agents, it

was not as important as the pelleting process. However, the authors also pointed out in their discussion

that a partial degradation of AFB1 by heat treatments had been reported by others, but this effect did

not occur in their study.

Commonly encountered discrepancies between in vitro and in vivo results for mycotoxin

binders outline the need of further research on the fate of the complex of mycotoxin with sequestering

agent in the gastrointestinal tract [43, 48]. Not only the effectiveness but also safety aspects of the

adsorbent materials for animals have been investigated in some studies [47].

Most of the research, however, deals with the efficacy of sequestering materials to bind

mycotoxins in vitro and in vivo. Methods used to study the effects of mycotoxin binders basically

include adsorption from aqueous solution, gastro-intestinal models and in vivo studies with different

animals. Recently, some extraction studies were performed to investigate aflatoxin B1 adsorption by

clays from water and corn meals [26]. It was shown that clays and activated carbon retained less AFB1

from aqueous corn meal dispersions than from water dispersions alone. It should be pointed out that

AFB1 was extracted from the aqueous corn meal dispersions with 60% methanol.

In vitro studies do not always predict in vivo results. Adsorption in vivo is complicated by

physiological variables and the composition of feed; factors which are rarely accounted for in vitro.

Apart from that, animal studies are costly and not easy to perform. However, all potential mycotoxin

binding agents must be tested in vivo to confirm their efficacy and safety and lack of interactions with

nutrients. An approach for an effective pre-screening mycotoxin/sorbent combinations before testing

on animals has been suggested [50]. The in vitro testing system developed so far, involved several

investigations:

(1) aqueous binding studies including single concentration sorption studies (isothermal);

(2) studies with the use of a gastrointestinal model;

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(3) modified isotherms to compare adsorption in the presence and absence of matrix.

The suggested approach allows the selection of the most promising materials as potential

enterosorbents for in vivo testing.

Several requirements have been suggested to assess mycotoxin binder performance in vivo.

These requirements include the following criteria: availability of essential nutrients to an animal; a

binder should not be a growth promoter (growth promotion may mask mycotoxins); improvement of

zootechnical performance; recovery of organ status; excretion of mycotoxins via faeces; broad-

spectrum mycotoxin binding performance; recovery of the immune status [51].

Analytical aspects

In response to the risk of great economic losses in the industry and the threat to human and

animal health as a result of exposure to mycotoxins, various analytical techniques have been developed

for their detection [52-55]. Commonly used methods include thin layer chromatography (TLC), high-

performance liquid chromatography (HPLC), gas chromatography (GC), and immunochemical

methods such as enzyme-linked immunosorbent assays (ELISA). In addition to the ELISA format,

rapid screening tests and a number of new techniques such as biosensors are rapidly emerging.

However, chromatographic techniques are usually used as reference methods for mycotoxin analysis.

The last few years have brought an incredible amount of information about binders and adsorbents

which can be used to decrease the bioavailability of mycotoxins. Various studies concerning the

performance of mycotoxin binding agents in vitro and in vivo have been carried out. To our

knowledge, no investigation regarding the influence of mycotoxin binders on the analytical

characteristics of the methods used for mycotoxin determination was performed. However, this issue is

of utmost importance, especially with respect to the authorisation of mycotoxin binding agents. Any

interference of mycotoxin binders with the analytical performance of a method can lead to incorrect

analytical findings and, as a consequence, to a misclassification with respect to the acceptance or

rejection of the material. As a result, the European Commission’s Directorate-General for Health and

Consumers (DG SANCO) asked the JRC to study the influence of mycotoxin binders on the

performance of mycotoxin testing methods. The background for this study is that binders shall not be

used to mask any previously non-compliant consignments as compliant by the addition of a binder.

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Outline of the Study

The aim of the present research was to investigate the possible effect of mycotoxin binding

agents on the analytical performance of standardised methods currently used for mycotoxin

determination in feed. The study on the influence of the binders on the analytical results was

performed for the following mycotoxins: AFB1, OTA, FUM B1 + B2, ZEA, DON, T2 + HT2 toxins.

Naturally contaminated or spiked feed materials were applied for the investigation. Not sufficiently

high naturally contaminated feed materials or materials with no corresponding mycotoxin found were

spiked to obtain levels approximately 1.5 times higher than the legislative or recommended limits. This

was the case for AFB1, DON and ZEA.

Mean values obtained for the untreated samples and the binder treated samples were compared

by performing Student’s t-test. The used variance was derived from the overall method repeatability.

These values are based on the method validation data obtained during the collaborative trial studies for

each method (Table 1). Differences were assumed to be significant at p < 0.05.

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Methodology

The test samples of commercial mycotoxin inactivation agents (mycotoxin binders) were

collected from various companies. Most of the test samples were obtained via the EU Feed Additives

and Premixtures Association (FEFANA). Three companies (Impextraco NV, Belgium; Sud-Chemie

AG, Germany; Agromed Austria GmbH, Austria) provided their samples independently. The samples

were coded with numbers to preserve the confidentiality of the source (Table 2). In some cases, the

identity of the producer/vendor was not disclosed. Type of the commercial products (main

components) and the range of their use in feed were communicated by FEFANA, the supplying

companies, or were stated in the commercial leaflets.

All samples were tested for pH in aqueous solutions, and infra-red spectra were recorded for

identification and characterisation purposes.

For pH measurement, a method for pH measurement of soils [56] was slightly modified to take

into account that some binders had a tendency to form stiff suspensions. Briefly, 1.0 g of a test sample

was suspended in 12.5 ml Milli-Q water. The pH-value was determined after a period of 60 min

directly in the suspension.

Infra-red (IR) spectroscopy studies were performed for the characterisation and identification

of the test material. This work has been done in collaboration with the Walloon Agricultural Research

Centre (see Annex 2).

Spiking solutions were prepared using mycotoxins from Sigma (Bornem, Belgium). Their

concentration was verified by the corresponding chromatographic method. Mycotoxin standard

solutions from Biopure (Tulln, Austria) were used for the calibration.

For spiking, 10.0 grams of dry ground feed material was fortified with 0.3 ml of the mycotoxin

solution in methanol (for AFB1) or in acetonitrile (for DON and ZEA). The samples were incubated

overnight in the dark at room temperature. A binder (or in case of grouped binder experiments, a

combination of different binders) was then mixed with the test material prior extraction. Maximum

recommended amounts of the mycotoxin binding agents were used in the experiments (Table 2). Feed

material without binder was used as a reference. The samples (with and without binders) were

prepared as independent triplicates for each experiment and each replicate was injected twice. The

samples were analysed according to the method protocol indicated below.

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For the experiments with AFB1 and DON, each binder was mixed with a portion of feed test

material (in triplicate). The whole set of analyses covering all binders was performed in several

batches. Feed material without binder was also analysed in triplicate in parallel with each batch.

For the experiments with OTA, ZEA, T-2 & HT-2 toxins, and fumonisins B1 + B2, the

mycotoxin binders were combined in groups according to their type. Thus, group A and group B were

aluminosilicate clays. Group A contained samples 2, 3, 4, 12, 13, and group B – samples 15-19. Group

C comprised yeast cell walls and included samples 1, 5, 6, 7 and 9. Group D included binders 8, 10,

11, which were mixtures of organic and mineral components. Group E was formed by fibre samples 14

and 20. For the above mentioned mycotoxins, all binders belonging to the same group were mixed

with a portion of the test sample (in triplicate). The feed material without binder was analysed in

triplicate as a reference.

Table 1. Repeatability data for each method based on collaborative trial data

Mycotoxin Average RSDr (%)

DON 10.6

AFB1 6.9

ZEA 7.7

T2 & HT2 toxins 9.8

FUM B1 + B2 3.2

OTA 4.1

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Table 2. Mycotoxin detoxifying agents and their amounts used in the study

Code

Type of main product(s)

pH value

Amount per 10 g

feed material

(mg)

1 Yeast cell wall 2.9 20

2 Clay + organic acid 7.9 10

3 Clay 8.4 10

4 Clay 4.6 25

5 Yeast cell wall 6.2 20

6 Yeast cell wall 6.7 20

7 Yeast cell wall 4.7 25

8 Mixture organic + mineral 6.2 25

9 Yeast cell wall 5.0 20

10 Mixture organic + mineral component 8.6 25

11 Mixture organic + mineral component 6.9 25

12 Clay 9.1 50

13 Clay 8.9 100

14 Fibres 6.7 100

15 Montmorillonite 10.4 40

16 Montmorillonite 10.6 40

17 HSCAS 8.8 50

18 HSCAS 9.8 50

19 HSCAS 9.8 50

20 Fibres (lignocellulose) 5.1 250

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Methods applied for the determination of mycotoxins

• Aflatoxin B1 has been determined following the standard EN ISO 17375:2006, 'Animal

feeding stuffs – determination of aflatoxin B1'.

• Deoxynivalenol has been determined using the method protocol of Stroka et.al. [57], which is

currently under consideration as a CEN standard (prEN 15791:2009).

• Zearalenone has been determined using the method protocol of Arranz et.al. [58], which is

currently under consideration as a CEN standard (prEN 15792:2009).

• Ochratoxin A has been determined using the method protocol of Stroka et.al. [59], which is

currently under consideration as a prospective CEN standard (working document N954 of

TC327/WG1).

• Fumonisins B1 and B2 have been determined using the method protocol of Breidbach et.al.

[60], which is currently under consideration as a prospective CEN standard (working document

N953 of TC327/WG1).

• T2 and HT2 toxins have been determined using the method protocol of Breidbach et.al. [61],

which is currently under consideration as a prospective CEN standard.

Data analysis

The data were analysed by performing Student’s t-test (independent two-sample t-test with

unequal sample sizes and equal variance). The used variance was derived from the overall method

repeatability. These values are based on the method validation data obtained during the collaborative

trial studies for each method (Table 1). Differences were assumed to be significant at p < 0.05.

Critical t-values (p = 0.05) were compared with those obtained by the Student’s t-test according to the

following formula:

21

11

21

nnS

XXt+×

−= ,

where

S – average repeatability relative standard deviation from collaborative trail (%)

X 1 - mean value determined for binder free material (set as 100 %)

18

X 2 - mean value determined for the test material with the binder added, relative to X 1 (%)

n1 – number of replicates for binder free material

n2 – number of replicates for the material containing mycotoxin binder

19

Results and Discussion

Aflatoxin B1 is currently the only mycotoxin for which legislative limits in feed materials are

set by the EC [10]. Most studies concerning the effects of mycotoxin binders are focused on AFB1.

Indeed, the great majority of binding agents, especially mineral adsorbents, are very effective in

alleviating aflatoxicosis but their efficacy against other mycotoxins is limited. Most mycotoxins are

extracted with an aqueous organic solvent. For example, AFB1 is extracted with a mixture of acetone-

water, 85:15 (v/v). This extraction solvent has the highest organic content compared to other solvents

used for the extraction of mycotoxins under current investigation. On the other hand, DON is the only

mycotoxin which requires the extraction with pure water for the method used in this study [57]. It can

be assumed that the mycotoxin binders perform most effectively in neat aqueous solutions. Thus, they

should have, if any, the strongest effect in the extraction solvent like it is used for DON. This

assumption is also supported by the fact that for the purification of mycotoxins in aqueous organic

solvents (e.g. acetonitrile/water), several methods for the determination of mycotoxins make use of

substances similar to the mycotoxin binders. These clean-up applications are based on the principle

that the purification agents used (e.g. charcoal or Celite®) do not bind to the mycotoxins, but retain

interfering co-extracted compounds [62-69]. Thus, they must have no or an insignificant effect on the

binding of mycotoxins from extracts with higher organic content. Other authors reported higher

retention of AFB1 by some clays from aqueous corn meal dispersions than from water, when 60%

methanol was used to extract AFB1 from the corn meal dispersions [26]. Taking into account these

aspects, AFB1 and DON were considered as two 'extreme' cases in our study. The effect of mycotoxin

binding agents on the analytical determination of these two mycotoxins was investigated extensively

for each single mycotoxin binder. For other mycotoxins (OTA, ZEA, FUM B1+B2, T-2 + HT-2), the

binders were grouped as described above and added together as a group to the test samples for the

extraction. This approach allows minimising the amount of experiments to a reasonable sequence

which can be carried out in one run for each mycotoxin. Moreover, it can enhance the effect of similar

binders.

For data analysis, critical t-values (p = 0.05) were compared with those obtained by the

Student’s independent two-sample t-test and applying the repeatability of each method based on

collaborative trial data (Table 1). The value corresponding to the method repeatability is a robust

estimate of the average performance of the standardised methods when applied in technically

competent laboratories. The repeatability of the methods as applied in our laboratory was in all cases

lower than the repeatability standard deviation as given in the CEN standards. This fact demonstrates

the laboratory's ability of applying the standard methods correctly.

20

As mentioned in the introduction, the method of addition (here pelleting) of a mycotoxin binder

was found to have an effect on the bioavailability of AFB1. It resulted in a reduced excretion of AFM1

to milk when animals were fed with AFB1 contaminated feed [49]. This aspect could not be

investigated in this study because of the unavailability of adequate processing machinery. The authors

of the above mentioned study claimed that feed processing had an effect only on the functionality of

the binder in the animal, and that the reduction of AFM1 in milk was not due to any degradation of

AFB1 during the processing. However, such a conclusion can only be made if the analytical method

used allows the complete recovery of the mycotoxin from the tested material.

Analysis of aflatoxin B1

No aflatoxin containing feed material was available for this study. Therefore, blank feed

material was spiked at 42 µg/kg as described above. Each binder was added in triplicate (Table 2). The

whole set of analyses covering all binders was performed in several batches. Spiking was carried out

for each analytical batch. The mycotoxin values obtained for the feed material without binder was used

as a reference and defined as 100 %. The results are presented in Figure 1. Statistical analysis indicated

that there were no significant differences between binder free material and the material containing

different binders under the described mixing conditions. Thus, it could be concluded that the addition

of the tested binders had no significant effect on the performance of the analytical procedure used for

the detection of AFB1.

Analysis of deoxynivalenol

Naturally contaminated feed material was used for these experiments. The amount of DON

found in this material was 6000 µg/kg. The material was additionally spiked at 6000 µg/kg to aim at a

total amount of DON of approximately 12000 µg/kg. This is approximately 1.5 times higher than the

recommended limit for animal feed based on cereals and cereal products with the exception of maize

by-products. The experimental design was the same as for AFB1. The results are given in Figure 2.

Statistical analysis indicated that there were no significant differences between binder free material

and the material containing different binders under the described mixing conditions. Thus, it could be

concluded that the addition of the tested binders had no significant effect on the performance of the

analytical procedure used for the detection of deoxynivalenol.

21

Binder

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Ret

rieva

l of a

flato

xin

B1,

%

0

20

40

60

80

100

120

Figure 1. Effect of different binders on the analytical determination of AFB1 by HPLC. The amount of AFB1 determined is related to that in binder free material defined as 100%. Bars are the mean values of triplicates. Error bars correspond to the repeatability (RSDr) of the method. Blue lines represent the RSDr range for binder free material. Binders were analysed in different batches indicated by spaces in the figure.

Binder

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Ret

rieva

l of d

eoxy

niva

leno

l, %

0

20

40

60

80

100

120

Figure 2. Effect of different binders on the analytical determination of DON by HPLC. The amount of DON determined is related to that in binder free material defined as 100%. Bars are the mean values of triplicates. Error bars correspond to the repeatability (RSDr) of the method. Blue lines represent the RSDr range for binder free material. Binders were analysed in different batches indicated by spaces in the figure.

22

Analysis of zearalenone

The same material used for the AFB1 experiments was used for the investigation concerning

ZEA. The material was found to be free of any naturally occurring ZEA. It was spiked at 2900 µg/kg.

For these experiments, binders were combined into groups as described in the section Methodology.

The binders, which belonged to the same group, were added together to each portion of feed test

material. All the binders were added to the test sample portions at the maximum recommended level

(Table 2). The feed material without binder was analysed as a reference and the level found was

defined as 100%. The results are presented in Figure 3. Statistical analysis indicated that there were no

significant differences between binder free material and the material containing different binders under

the described mixing conditions. Thus, it could be concluded that the addition of the tested binders had

no significant effect on the performance of the analytical procedure used for the detection of ZEA.

Binder group

A B C D E

Ret

rieva

l of z

eara

leno

ne, %

0

20

40

60

80

100

Figure 3. Effect of different binders on the analytical determination of ZEA by HPLC. The amount of ZEA determined is related to that in binder free material defined as 100%. Bars are the mean values of triplicates. Error bars correspond to the repeatability (RSDr) of the method. Blue lines represent the RSDr range for binder free material.

23

Analysis of ochratoxin A

For the investigation of mycotoxin binder effects for OTA, naturally contaminated feed

material was used. The amount of OTA in binder free material has been determined as 367 µg/kg. The

experiment was designed in the same way as for ZEA (addition of binders by groups). The results are

given in Figure 4. Statistical analysis indicated that there were no significant differences between

binder free material and the material containing different binders under the described mixing

conditions. Thus, it could be concluded that the addition of the tested binders had no significant effect

on the performance of the analytical procedure used for the detection of OTA.

Analysis of fumonisins B1+B2

For the investigation of mycotoxin binder effects concerning fumonisins, naturally

contaminated feed material was used. The total amount of FUM B1 and B2 in the binder free material

has been determined as 7900 µg/kg. The experiment was designed in the same way as for ZEA and

OTA (addition of binders by groups). The results are shown in Figure 5. Statistical analysis indicated

that there were no significant differences between binder free material and the material containing

different binders under the described mixing conditions. Thus, it could be concluded that the addition

of the tested binders had no significant effect on the performance of the analytical procedure used for

the detection of fumonisins B1 and B2.

Analysis of T-2 + HT-2 toxins

For the investigation of mycotoxin binder effects concerning T-2 and HT-2 toxins, naturally

contaminated feed material (same as for DON experiments) was used. The experiment was designed in

the same way as for ZEA, OTA and the fumonisins (addition of binders by groups). The total amount

of T2 and HT-2 toxins in the binder free material has been determined as 7850 µg/kg. The results are

given in Figure 6. Statistical analysis indicated that there were no significant differences between

binder free material and the material containing different binders under the described mixing

conditions. Thus, it could be concluded that the addition of the tested binders had no significant effect

on the performance of the analytical procedure used for the detection of T-2 and HT-2 toxins.

24

Binder group

A B C D E

Ret

rieva

l of o

chra

toxi

n A

, %

0

20

40

60

80

100

Figure 4. Effect of different binders on the analytical determination of OTA by HPLC. The amount of OTA determined is related to that in binder free material defined as 100%. Bars are the mean values of triplicates. Error bars correspond to the repeatability (RSDr) of the method. Blue lines represent the RSDr range for binder free material.

Binder group

A B C D E

Ret

rieva

l of f

umon

isin

s B

1 & B

2, %

0

20

40

60

80

100

Figure 5. Effect of different binders on the analytical determination of FUM B1+B2 by HPLC. The amount of FUM B1 + B2 determined is related to that in binder free material defined as 100%. Bars are the mean values of triplicates. Error bars correspond to the repeatability (RSDr) of the method. Blue lines represent the RSDr range for binder free material.

25

Binder group

A B C D E

Ret

rieva

l of T

-2 &

HT-

2 to

xins

, %

0

20

40

60

80

100

120

Figure 6. Effect of different binders on the analytical determination of the total amount of T-2 and HT-2 toxins by GC-MS. The amount of T-2 and HT-2 toxins determined is related to that in binder free material defined as 100%. Bars are the mean values of triplicates. Error bars correspond to the repeatability (RSDr) of the method. Blue lines represent the RSDr range for binder free material.

26

Conclusions

The effect of mycotoxin binders in animal feed on the analytical performance of currently standardised

methods for the determination of mycotoxins in feed was investigated. Statistical analysis did not show

any significant differences in the selected analytical methods’ capacity to determine mycotoxins in

binder free material and materials with mycotoxin binders added, respectively. Thus, it could be

concluded that the tested binders had no effect on the level of mycotoxins found. It is therefore not

possible to use any of the tested binders for masking mycotoxins in contaminated feed under the

condition that the binders are used within the recommended range, and that they are mixed into the

feed in the same manner as in our study. A combination of binder addition and processing, such as

pelleting or extrusion, has not been a subject of this study but could be considered as a next step.

27

Acknowledgement

We are grateful to Mr. D. Jans (FEFANA) and Mr. G. Bertin (Alltech) for providing us with the test

samples of mycotoxin binders collected from various companies.

We would like also to acknowledge Mr. T. Vanderborght (Impextraco NV), Mr. A. Kroismayr

(Agromed Austria GmbH) and Sud-Chemie AG for providing the test samples of mycotoxin binders.

We are grateful to Dr. V. Baeten, Dr. O. Abbas and Mr. A. Rodriguez (Walloon Agricultural Research

Centre) for the spectroscopy studies on mycotoxin binders.

We also wish to thank Dr. C. Von Holst and Mr. B. Slowikowski (JRC-IRMM) for their support in this

research.

The authors wish to thank Anne-Mette Jensen for valuable comments.

28

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trichothecenes. Talanta 73, 251–257.

65. Ren, Y., Zhang, Y., Shao, S., Cai, Z., Feng, L., Pan, H., Wang, Z., 2007. Simultaneous

determination of multi-component mycotoxin contaminants in foods and feeds by ultra-

performance liquid chromatography tandem mass spectrometry. J. Chromatogr. A, 1143, 48–

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66. Schothorst, R.C., Jekel, A.A., Van Egmond, H.P., De Mul, A., Boon, P.E., Van Klaveren, J.D.,

2005. Determination of trichothecenes in duplicate diets of young children by capillary gas

chromatography with mass spectrometric detection. Food Addit. Contam. 22, 48–55.

67. Valle-Algarra, F.M., Medina, A., Gimeno-Adelantado, J.V., Llorens, A., Jimenez, M., Mateo,

R., 2005. Comparative assessment of solid-phase extraction clean-up procedures, GC columns

and perfluoroacylation reagents for determination of type B trichothecenes in wheat by GC–

ECD. Talanta, 194–201.

68. Krska, R., 1998. Performance of modern sample preparation techniques in the analysis of

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69. Silva, C.M.G., E. Vargas, E.A., 2001. A survey of zearalenone in corn using Romer

MycosepTM 224 column and high performance liquid chromatography. Food Addit. Contam.

18, 39-45.

34

Annex 1

Results of the determination of mycotoxins in feed samples by the standardised methods with and

without mycotoxin binders

t-value Amount of mycotoxin, µg/kg Mycotoxin, batch

Binder(s) added critical calculated Prep. 1 Prep. 2 Prep. 3

Blank - - 38.52 39.55 38.71 Binder 1 2.78 0.31 38.57 38.21 37.95 Binder 2 2.78 0.05 38.75 38.74 39.62 Binder 3 2.78 0.18 40.81 37.63 39.52

AFB1 Batch 1

Binder 4 2.78 0.26 38.25 38.26 38.58 Blank - - 37.76 39.84 36.95 Binder 5 2.78 0.99 37.71 37.61 45.59 Binder 6 2.78 0.34 39.78 39.37 37.62 Binder 7 2.78 0.08 39.43 37.44 37.18 Binder 8 2.78 0.76 37.65 34.46 37.55 Binder 9 2.78 0.39 38.51 37.05 36.46

AFB1 Batch 2

Binder 10 2.78 0.29 37.99 40.12 38.28 Blank - - 40.35 39.87 40.54 Binder 11 3.18 0.05 - 39.44 40.81 Binder 12 2.78 0.28 39.17 39.75 39.92 Binder 13 2.78 0.00 40.40 41.07 39.32 Binder 14 3.18 0.15 40.15 - 39.61

AFB1 Batch 3

Binder 15 2.78 0.17 39.48 40.82 39.34 Blank - - 39.23 39.71 39.64 Binder 16 3.18 0.50 - 38.15 38.44 Binder 17 2.78 0.50 38.35 38.21 38.69 Binder 18 2.78 0.61 38.07 38.35 38.09 Binder 19 2.78 0.06 38.91 39.32 39.94

AFB1 Batch 4

Binder 20 2.78 0.19 39.20 38.94 39.18 Blank - - 10234 10116 10017 Binder 1 2.78 0.02 10355 10230 9832 Binder 2 2.78 0.62 9668 9172 9895 Binder 3 2.78 0.82 10097 8996 9120 Binder 4 2.78 0.70 9747 9233 9553

DON Batch 1

Binder 5 2.78 0.02 10114 10308 9880 Blank - - 10362 10668 9982 Binder 6 2.78 0.37 9958 9651 10418 Binder 7 2.78 0.73 10235 9324 9485 Binder 8 3.18 0.24 11465 - 9700 Binder 9 2.78 0.30 10153 10160 9878

DON Batch 2

Binder 10 2.78 1.51 8218 8862 9856 Blank - - 10154 10451 8734 Binder 11 2.78 0.11 10239 9893 8915 Binder 12 2.78 0.83 8414 8406 10416 Binder 13 2.78 0.59 9307 9205 9314 Binder 14 2.78 0.31 9391 9823 9336

DON Batch 3

Binder 15 2.78 0.30 9630 9437 9505

35

t-value Amount of mycotoxin, µg/kg Binder(s) added critical calucated Prep. 1 Prep. 2 Prep. 3

Blank - - 9740 9805 10023 Binder 16 2.78 0.23 9430 9634 9907 Binder 17 2.78 0.35 9572 9594 9513 Binder 18 2.78 0.09 9480 9747 10111 Binder 19 2.78 0.43 9596 9505 9366

DON Batch 4

Binder 20 2.78 0.43 9541 9577 9339 Blank - - 2716 2777 2778 Group A 2.78 0.05 2843 2753 2699 Group B 2.78 0.02 2889 2611 2759 Group C 3.18 0.18 - 2735 2849 Group D 2.78 0.55 2811 2928 2818

ZEA

Group E 2.78 0.09 2702 2870 2650 Blank - - 370.2 368.6 361.5 Group A 2.78 0.75 371.9 376.9 379.3 Group B 2.78 0.66 365.3 384.4 375.3 Group C 2.78 0.27 369.4 373.4 367.7 Group D 2.78 0.57 372.5 365.7 383.2

OTA

Group E 2.78 0.15 371.4 353.9 369.3 Blank - - 7955 7822 7923 Group A 2.78 0.93 7710 7800 7618 Group B 2.78 0.66 7790 7834 7667 Group C 2.78 1.06 7736 7730 7581 Group D 2.78 1.57 7651 7705 7377

FUM B1 + B2

Group E 2.78 1.05 7643 7623 7788 Blank - - 7705 7690 8165 Group A 2.78 0.00 7795 8065 7705 Group B 2.78 0.20 7805 7725 7655 Group C 3.18 0.18 - 7625 7825 Group D 2.78 0.13 7830 7790 7705

T2 + HT2

Group E 2.78 0.30 7635 7655 7710

36

Annex 2

(see next page)

37

Infrared Measurements on Mycotoxin Binders

Report prepared by

Ouissam Abbas and Vincent Baeten In collaboration with Alexandro Rodriguez

Walloon Agricultural Research Centre (CRA-W)

Chaussée de Namur 24

B-5030 Gembloux, Belgium

Working document from 17 July 2009 (including the work performed during the

period of April – June 2009)

Walloon Agricultural Research Centre Quality of Agricultural Products Department Chaussée de Namur, 24 B – 5030 GEMBLOUX Tél :++ 32 (0) 81/62.03.50 Fax : ++ 32 (0) 81/62.03.88 baeten@cra.wallonie.be

http://cra.wallonie.be

Wal

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38

Contents

1 Summary ......................................................................................................................... 39

1.1 Framework of the study............................................................................................ 39

1.2 Objectives ................................................................................................................. 39

2 Material and Methods.................................................................................................... 39

2.1 Samples..................................................................................................................... 39

2.2 Instruments ............................................................................................................... 41

2.3 Methodology............................................................................................................. 41

3 Results and discussion.................................................................................................... 41

3.1 Yeast cell wall........................................................................................................... 42

3.2 Clay .......................................................................................................................... 43

3.3 Mixture of organic and mineral components ........................................................... 43

3.4 Montmorillonite........................................................................................................ 44

3.5 Hydrated Sodium Calcium Aluminosilicate (HSCAS).............................................. 45

3.6 Additional reference substances............................................................................... 45

4 Conclusions ..................................................................................................................... 46

39

1 Summary

The purpose of this work is to check if the different materials from the same group of substances are identical

and to characterise their identity. Spectroscopic methods (Raman, mid and near infrared) are used to have

additional and complementary information.

Due to the problems encountered with mid infrared and Raman methods, only near infrared results are

presented in this report.

1.1 Framework of the study

This work is carried out in the framework of the study asked by the European Commission, DG Joint Research

Centre (Institute for Reference Materials and Measurements) in order to use spectroscopic methods to identify

mycotoxin binders.

1.2 Objectives

The objectives aimed by this work were as follows:

• The characterization of mycotoxin binders

• The use of spectroscopy to investigate if the different materials from the same group of substances are

identical

• To build a small database of different mycotoxin binder classes which allows to check, if needed in

future studies, whether there is an evolution of material composition of available mycotoxin binders over

time

2 Material and Methods

2.1 Samples

27 samples were measured by Raman and near infrared spectrometry (Tables 1 and 2).

40

Table 1. Studied mycotoxin binders

Code Product

DQ09-0350-001 Yeast cell wall

DQ09-0350-002 Clay + organic acid

DQ09-0350-003 Clay

DQ09-0350-004 Clay

DQ09-0350-005 Yeast cell wall

DQ09-0350-006 Yeast cell wall

DQ09-0350-007 Yeast cell wall

DQ09-0350-008 Mixture organic + mineral

DQ09-0350-009 Yeast cell wall

DQ09-0350-010 Mixture organic + mineral component

DQ09-0350-011 Mixture organic + mineral component

DQ09-0350-012 Clay

DQ09-0350-013 Clay

DQ09-0350-014 Fibres

DQ09-0350-015 Montmorillonite

DQ09-0350-016 Montmorillonite

DQ09-0350-017 HSCAS

DQ09-0350-018 HSCAS

DQ09-0350-019 HSCAS

DQ09-0350-027 Fibres (lignocellulose)

Table 2. Additional reference substances studied

Code Product

DQ09-0350-020 Florisil

DQ09-0350-021 Kieselgur

DQ09-0350-022 Bentonite

DQ09-0350-023 Zeolite

DQ09-0350-024 Activated charcoal

DQ09-0350-025 Sepiolite

DQ09-0350-026 Dowex

41

2.2 Instruments

Raman analyses were performed using RAMII vertex 70 spectrometer (Bruker).

Near infrared spectroscopic analyses were performed using NirSystem spectrometer (Foss).

Software: OPUS (version 6.5), WinIsi III and The Unscrambler.

2.3 Methodology

The analytical methods followed are presented in the Table 3.

Table 3. Experimental conditions for spectra recording

Raman Near infrared Mid infrared

Range 3600 - 0 cm-1 400 – 2498 nm

Resolution 4 cm-1 2 nm

Number of scans 128 scans 32 scans

Spectra were

collected as

Raman

intensities

Spectra were

collected as log

1/R

Problems encountered

with ATR accessory

No measurements

could be done

3 Results and discussion

Three spectroscopic methods were tested on the mycotoxins binders. However when using the MIR instrument,

the Attenuated Total Reflectance (ATR) accessory failed. Therefore, no mid-infrared spectra could be

generated.

Raman analyses were conducted but spectra obtained were not well resolved. This made it difficult to make

interpretations in order to characterize studied samples (data not shown).

As a result only near infrared method was able to give the spectra with good quality.

Because of the use of only one method (NIR), the objective concerning the characterization of mycotoxin

binders couldn’t be reached. In fact, to have precise and complete information about the composition of the

studied samples, two or three methods (NIR/MIR, NIR/Raman or MIR/Raman) should be combined.

Near infrared spectra were used to see if, at a spectroscopic level, the different materials from the same group

of substances were identical. CRA-W will proceed by the comparison of spectra of the same family; we will

regroup spectra according to their similarity. In fact, the same spectral profile indicates similar composition. Six

groups will be studied in this report: yeast cell wall, clay, mixture of organic and mineral components,

montmorillonite, hydrated sodium calcium aluminosilicate (HSCAS) and additional reference substances.

42

3.1 Yeast cell wall

Spectra of yeast cell wall exhibit some differences and they are regrouped by pairs (a, b) (Figure 1).

Spectra are regrouped as presented in the table below.

a DQ09-0350-001 Yeast cell wall 500 g-2 kg/T feed DQ09-0350-007 Yeast cell wall 0,5 - 2,5 kg/T feed b DQ09-0350-005 Yeast cell wall 1-2 kg/T feed DQ09-0350-006 Yeast cell wall 1-2 kg/T feed

The spectra have the same bands but differences are present in 492-640 nm and 2008-2230 nm regions. This

indicates some difference in the composition of these sub-groups of yeast cell wall.

Figure 1: NIR spectra of yeast cell wall.

The region below 1100 nm will be excluded because for the rest of the samples, no differences between spectra

occur in this region.

43

3.2 Clay

The spectra generated between 1100 and 2498 nm are compared as shown in figure 2. Five clay samples

seem to be different. However, there are some similarities between spectra of samples DQ09-0350-02 and

DQ09-0350-04 (a) and of samples DQ09-0350-03 and DQ09-0350-12 (b). One band is absent in each case:

between 2272 and 2330 nm for sample DQ09-0350-04 and between 2370 and 2436 nm for sample DQ09-0350-

12. Sample DQ09-0350-13 has totally different spectrum compared to others indicating another composition.

This sample has the highest level of use (2-10 kg/T feed).

Figure 2: NIR spectra of clay.

3.3 Mixture of organic and mineral components As shown in figure 3, spectra of three samples of mixtures of organic and mineral components have the same

band between 1860 and 2024 nm but the band of the samples DQ09-0350-11 is slightly shifted and has

maximum at 1920 nm while two other spectra have maximum at 1910 nm. The region of the higher wavelengths

(> 2024 nm) shows more dissimilarity. Differences in the composition (other proportions or different

components) can be assumed.

44

Figure 3: NIR spectra of mixtures of organic and mineral components.

3.4 Montmorillonite

Figure 4: NIR spectra of montmorillonite.

45

Figure 4 indicates that the spectra of two samples of montmorillonite (DQ09-0350-15 and DQ09-0350-16) are

different. Differences occur in the regions 1684-1770 nm and 2278-2378 nm.

3.5 Hydrated Sodium Calcium Aluminosilicate (HSCAS)

Spectra of the hydrated sodium calcium aluminosilicate (HSCAS) shown in figure 5 are very similar. However,

for sample DQ09-0350-19, the bands in the region 2164-2230 nm are missing. This compound can be

considered as similar to two other samples (DQ09-0350-17 and DQ09-0350-18), with some minor differences.

Figure 5: NIR spectra of hydrated sodium calcium aluminosilicate (HSCAS).

3.6 Additional reference substances

For those samples, spectra are given without any comparison (Figure 6). It can be noted from figure 6 that

each compound has its own spectral profile. Bands present at the appropriate wavelengths inform on the

composition of each compound.

46

Figure 6: NIR spectra of the additional reference substances.

4 Conclusions

Comparison of near infrared spectra of compounds from the same group has shown that some differences can

be observed even for the samples from the same family. This indicates some additional components or

differences in proportions in the case of mixtures. Near infrared spectroscopy is an effective and rapid tool

which can be used for screening purposes and for the evaluation of the composition of a compound or a

mixture.

The authors believe that it is interesting to continue the work in the future to estimate the evolution of mycotoxin

binding materials in time.

European Commission EUR 23997 EN – Joint Research Centre – Institute for Reference Materials and Measurements Title: Evaluation of the Effect of Mycotoxin Binders in Animal Feed on the Analytical Performance of Standardised Methods for the Determination of Mycotoxins in Feed Author(s): A. Kolossova, J. Stroka, A. Breidbach, K. Kroeger, M. Ambrosio, K. Bouten, F. Ulberth Luxembourg: Office for Official Publications of the European Communities 2009 – 46 pp. – 21.0 x 29.7 cm EUR – Scientific and Technical Research series – ISSN 1018-5593 ISBN 978-92-79-13106-6 DOI 10.2787/15352 Abstract The last few years have brought a vast amount of information about mycotoxin inactivation agents (mycotoxin binders) which are applied with the aim to reduce the toxic effects of mycotoxins in animals. The influence of the addition of mycotoxin binders to animal feed on the analytical performance of the methods for the determination of mycotoxins was studied and the results are presented in this report. Standardised methods already available or currently under consideration at the European Standardization Committee (CEN) have been applied for the analysis of mycotoxins in feed materials. Samples of 20 commercial mycotoxin inactivation agents were collected from various companies. The following mycotoxins were included in the study: aflatoxin B1, deoxynivalenol, zearalenone, ochratoxin A, fumonisins B1 + B2, T2 and HT2 toxins. Naturally contaminated or spiked feed materials and the maximum recommended amounts of the mycotoxin detoxifying agents were used in the experiments. A binder (or binders combined in a group) was mixed with feed material containing the corresponding mycotoxin, and the feed material with and without binder was analysed using the appropriate method. For data evaluation, the obtained mean values were compared by Student’s t-test (independent two-sample t-test with unequal sample sizes and equal variance). The repeatability standard deviation of each method based on collaborative trial data was used as an estimate of method variability. No significant differences (p = 0.05) in mycotoxin levels between binder free material and the material containing different binders have been found under the applied conditions.

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